A naturally occurring Wolbachia strain (wAnga-Mali) was identified in mosquitoes of the Anopheles gambiae complex collected in the Malian villages of Dangassa and Kenieroba. Phylogenetic analysis of the nucleotide sequence of two 16S rRNA regions showed that wAnga-Mali clusters with Wolbachia strains from supergroup A and has the highest homology to a Wolbachia strain isolated from cat fleas (Ctenocephalides). wAnga-Mali is different from two Wolbachia strains previously reported in A. gambiae from Burkina Faso (wAnga_VK5_STP and wAnga_VK5_3.1a). Quantitative analysis of Wolbachia and Plasmodium sporozoite infection in field-collected mosquitoes indicates that the prevalence and intensity of Plasmodium falciparum sporozoite infection is significantly lower in Wolbachia-infected females. The presence of Wolbachia in females from a laboratory Anopheles coluzzii (A. gambiae, M form) colony experimentally infected with P. falciparum (NF54 strain) gametocyte cultures slightly enhanced oocyst infection. However, Wolbachiainfection significantly reduced the prevalence and intensity of sporozoite infection, as observed in the field. This indicates that wAnga-Mali infection does not limit early stages of Plasmodium infection in the mosquito, but it has a strong deleterious effect on sporozoites and reduces malaria transmission. This work was published in PNAS. We are currently investigating how the prevalence and intensity of Wolbachia infection varies thought the transmission season and in different villages. Different larval breeding sites in Dangassa and Kenieroba will also be evaluated to establish whether the level of infection with Wolbachia is homogeneous or varies depending on the mosquito breeding site. Transmission-blocking vaccines are based on eliciting antibody responses in the vertebrate host that disrupt parasite development in the mosquito vector and prevent malaria transmission. The surface protein Pfs47 is present in Plasmodium falciparum gametocytes and female gametes. The potential of Pfs47 as a vaccine target was evaluated. Soluble full-length recombinant protein, consisting of three domains, was expressed in E. coli as a thioredoxin fusion (T-Pfs47). The protein was immunogenic, and polyclonal and monoclonal antibodies (mAb) were obtained, but they did not confer transmission blocking activity (TBA). All fourteen mAb targeted either domains 1 or 3, but not domain 2 (D2), and immune reactivity to D2 was also very low in polyclonal mouse IgG after T-Pfs47 immunization. Disruption of the predicted disulfide bond in D2, by replacing cysteines for alanines (C230A and C260A), allowed expression of recombinant D2 protein in E. coli. A combination of mAbs targeting D2, and deletion proteins from this domain, allowed us to map a central 52 amino acid (aa) region where antibody binding confers strong TBA (78-99%). This 52 aa antigen is immunogenic and well conserved, with only seven haplotypes world-wide that share 96-98% identity. Neither human complement nor the mosquito complement-like system were required for the observed TBA. A dramatic reduction in ookinete numbers and ookinete-specific transcripts was observed, suggesting that the antibodies are interacting with female gametocytes and preventing fertilization. This work was published in Vaccines npg. We are currently collaborating with Dr. Patrick Duffy and Dr. Robert Seder to optimize the immunogenicity of this recombinant protein by conjugating it to different carriers and testing different adjuvant systems. We are also exploring the use of microneedles as a delivery system. The mosquito complement-like system is a major defense mechanism that limits Plasmodium infection. Ookinete midgut invasion results in irreversible damage to invaded cells and triggers epithelial nitration and complement activation. Several lines of evidence suggest that hemocytes participate in early antiplasmodial responses that target ookinetes, but their role remains unclear. The fate of hemocytes in response to Plasmodium infection was investigated by labeling this cell population in vivo. We found that midgut nitration triggers the local release of hemocyte-derived microvesicles (HdMv) into the basal labyrinth of the midgut. Several different strategies, such as gene silencing, immune priming, or systemic injection of polystyrene beads, were used to either enhance or reduce HdMv release. We provide direct experimental evidence that contact of hemocytes with the nitrated midgut basal surface triggers HdMv release and that this response is necessary for effective activation of mosquito complement. Our studies suggest that hemocyte-derived microvesicles may deliver some critical factor(s) that promote activation of thioester-containing protein 1, a key effector of the mosquito antiplasmodial immunity. This work was published in Science Immunology. The nature of the signals that attract hemocytes to the midgut epithelial cells invaded by the parasite was investigated. When ookinetes invade the midgut epithelium, they disrupt the barriers that normally prevent direct contact between bacteria from the gut microbiota and epithelial cells. We found that feeding mosquitoes a protein solution containing heat-killed bacteria triggers the release of prostaglandin by epithelial cells. It also attracts circulating hemocytes to the basal surface of the midgut, increases their patrolling activity and activates an immune priming response. Hemocytes also exhibit a strong chemotactic response to prostaglandin. Furthermore, ookinete invasion, in the presence bacteria from the microbiota, triggers a systemic release of prostaglandin and systemic injection of prostaglandin is sufficient to establish a priming response. The source of prostaglandins in insects has been a mystery, because they lack cyclooxygenase. We identified two-heme peroxidases that are induced my midgut cells in response to contact with bacteria and are essential for the biosynthesis of prostaglandin and to establish the priming response. The manuscript describing this work is under preparation.